It is clear that dopamine (DA) at high concentrations (> 100 nmol/l) inhibits the release of prolactin (PRL). Paradoxically, this monoamine at low concentrations (< 10 nmol/l) has also been shown to augment PRL secretion. One possible explanation for these divergent effects is that DA binds receptors capable of interacting with multiple G protein subtypes that recruit opposing intracellular signaling pathways within lactotropes. To identify G proteins which couple DA receptor activation to PRL secretion, we have selectively immunoneutralized the activity of Gi alpha 3 and Gs alpha in primary cultures of rat pituitaries and subsequently tested the ability of these cultures to respond to high and low dose DA. Specifically, permeabilized pituitary cell cultures from random-cycling female rats were treated with control immunoglobulins (IgGs; 50 micrograms/ml) purified from preimmune serum (PII) or IgGs directed against the C-terminal portion of Gi alpha 3 or Gs alpha. After immunoneutralization of these G proteins, cells were challenged with 10 or 1000 nmol DA/l and the relative amount of PRL released was assessed by reverse hemolytic plaque assay. Results were expressed as % of basal values and compared. Under control conditions (PII), 1000 nmol DA/l inhibited (61.4 +/- 7.6% of basal values; mean +/- S.E.M.) while 10 nmol DA/l augmented (120.0 +/- 7.0%) PRL release in five separate experiments. Treatment of cells with anti-Gi alpha 3 attenuated the inhibitory effect of high dose DA (87.3 +/- 14.5%). However, elimination of Gi alpha 3 activity did not significantly alter the PRL stimulatory effect of 10 nmol DA/l (121.0 +/- 5.2%). Interestingly, immunoneutralization of Gs alpha resulted in a reciprocal shift in the activity of the lower dose of DA from stimulatory to inhibitory (69.7 +/- 7.3%) while combined treatment of anti-Gi alpha 3 and anti-Gs alpha abrogated the responsiveness of pituitary cell cultures to either DA treatment (1000 nmol/l, 70.7 +/- 12.5% and 10 nmol/l, 87.5 +/- 21.4%). These data reveal that ligand-activated DA receptors can interact with both Gi alpha 3 and Gs alpha. Elimination of the stimulatory component (Gs alpha) favors the DA receptor activation of the inhibitory pathway (Gi alpha 3) suggesting a competition between negative and positive intracellular signaling mechanisms in normal lactotropes. In addition to DA treatment, we also challenged permeabilized pituitary cells with 100 nmol thyrotropin-releasing hormone (TRH)/l as a positive control for secretory integrity. As anticipated, TRH stimulated PRL release to 188.0 +/- 31.0% of basal values under control conditions. Unexpectedly, immunoneutralization of Gs alpha completely blocked the ability of TRH to induce PRL release (101.8 +/- 12.0%). This neutralizing effect was specific to Gs alpha in that blockade of Gi alpha 3 activity had no significant effect on TRH-stimulated PRL release (166.2 +/- 13.1%). These data are the first to support a direct role of Gs alpha in TRH signal transduction within PRL-secreting cells.